Biogenesis and assembly of eukaryotic multispanning integral membrane proteins is proposed to occur cotranslationally through the action of independent topogenic sequences which direct sequential translocation initiation, termination and membrane integration events at the ER membrane. Recent studies, however, have demonstrated new complexities in this biosynthetic pathway. For a subset of native multispanning proteins, cooperative interactions rather than sequential independent activities may transiently delay integration of the chain into the membrane until synthesis of multiple transmembrane segments has been completed. Remarkably, novel topogenic determinants have also been identified which direct biogenesis of multiple topologic isoforms from a single polypeptide chain. These findings have important implications for the structure and function of diverse membrane proteins and require a detailed reexamination of the molecular mechanisms involved in their assembly. To better understand different biogenesis pathways utilized by polytopic proteins, the proposed studies will: i) identify the nature of information encoded within sequence determinants in the nascent chain which direct different translocation and membrane integration events, ii) determine how this information is interpreted by translocation machinery and/or chaperones at the ER membrane to effect different assembly mechanisms and, iii) investigate the effects of different biogenesis pathways on final protein structure and function. Three different proteins, human MDR1, CHIP28, AND MIWC, each of which demonstrate different biogenesis pathways will be studied using cell-free translation systems, Xenopus oocytes and cultured mammalian cells. Topogenic sequence determinants will be characterized in defined protein chimeras and in native contexts to identify key information responsible for translocation specificity and membrane integration. Interactions between these determinants and components of the translocation machinery will be identified by crosslinking ER proteins to nascent chains at specific stages of biogenesis. Finally, the role of different topological isoforms in protein maturation and function will be investigated. These studies will accomplish three important goals. First, they systematically define determinants which direct distinct events of topological maturation. Second, they identify translocation machinery and cellular chaperones through which these determinants act. Third, they correlate different biogenesis mechanisms with requirements for protein function. This work will provide a detailed understanding of cellular mechanisms which regulate polytopic protein assembly and allow further investigation into how these pathways might be disrupted in acquired or inherited human diseases.